The guiding objective of the proposed research is to increase our understanding of the mechanism of action of dihydrofolate reductase (DHFR) in terms of its molecular structure. DHFR is the target of antifolate drugs such as methotrexate, trimethoprim and pyrimethamine, which are useful as antineoplastics and antibiotics. Detailed understanding of the structure of this molecule and how it functions should aid in efforts to design more sophisticated disease-specific inhibitors. Specific aims include determining the structure of folate (substrate) containing complexes of the enzyme from E. coli and chicken; refinement at high resolution of the R-67-plasmid DHFR; elucidation of the chicken enzyme structure when it is activated by mercurials; and to crystallize the R-plasmid enzyme in a complex with substrate and cofactor.